Successful production of recombinant proteins has been accomplished with eukaryotic hosts. The most prominent examples are yeasts like Saccharomyces cerevisiae, Pichia pastoris or Hansenula polymorpha, filamentous fungi like Aspergillus awamori or Trichoderma reesei, or mammalian cells like e.g. CHO cells. While the production of some proteins is readily achieved at high rates, many other proteins are only obtained at comparatively low levels.
The heterologous expression of a gene in a host organism usually requires a vector allowing stable transformation of the host organism. A vector would provide the gene with a functional promoter adjacent to the 5′ end of the coding sequence. The transcription is thereby regulated and initiated by this promoter sequence. Most promoters used up to date have been derived from genes that code for proteins that are usually present at high concentrations in the cell.
EP0103409A2 discloses the use of yeast promoters associated with expression of specific enzymes in the glycolytic pathway, i.e. promoters involved in expression of pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, phosphor-glycerate mutase, hexokinase 1 and 2, glucokinase, phosphofructose kinase, aldolase and glycolytic regulation gene.
WO 97/44470 describes yeast promoters from Yarrowia lipolytica for the translation elongation factor 1 (TEF1) protein and for the ribosomal protein S7 that are suitable for heterologous expression of proteins in yeast, and EP1951877A1 describes the use of the P. pastoris TEF1 promoter for the production of heterologous proteins.
WO2005003310 provides methods for the expression of a coding sequence of interest in yeast using a promoter of the glyceraldehyde-3-phosphate dehydrogenase or phosphoglycerate mutase from oleaginous yeast Yarrowia lipolytica. 
Promoter sequences derived from genes involved in the methanol metabolic pathway of Pichia pastoris are disclosed in U.S. Pat. Nos. 4,808,537 and 4,855,231 (alcohol oxidase AOX1, AOX2) and U.S. Pat. No. 6,730,499B1 (formaldehyde dehydrogenase FLD1). US20080153126A1 includes mutant promoter sequences based on the AOX1 promoter.
The AOX1 promoter is induced only in response to methanol and repressed by other carbon sources, such as glucose or ethanol. Methanol has the disadvantage that it is unsuitable for use in the production of certain products, since it is potentially hazardous for its toxicity and flammability. Therefore, alternatives to the AOX1 promoter are sought.
US2008299616A1 introduces the regulatory sequences of the malate synthase (MLS1) gene for heterologous gene expression in P. pastoris, which is repressed in media containing glucose and derepressed under glucose starvation conditions or when acetate is present. However, this system is not considered suitable for efficient production methods, since the MLS1 promoter is weak with low activity under de-repressed conditions.
Schöler and Schüller (Mol. Cell Biol. 1994 14(6):3613-22) describe the control region of the isocitrate lyase gene ICL1, which is derepressed after transfer of cells from fermentative to non-fermentative growth conditions.
WO2008063302A2 describes the use of novel inducible promoters derived from ADH1 (alcohol dehydrogenase), ENO1 (enolase) and GUT1 genes of P. pastoris for the expression of heterologous proteins, CN1966688A the P. pastoris omega 3-fatty acid dehydrogenase promoter sequence, and WO002007117062A1 the P. pastoris derived auto-inducible NPS promoter, which is induced by phosphor limitation.
WO2008128701A2 describes the use of novel promoters, of which the promoter derived from the THI3 (thiamine metabolism) gene of P. pastoris is repressed in medium containing thiamine, and derepressed upon thiamine depletion.
US2009325241A1 describes a method of ethanol production in a yeast cell employing a xylose-inducible promoter (FAS2 promoter).
It is desirable to provide improved recombinant eukaryotic cell lines to produce fermentation products that can be isolated with high yields. Therefore, it is the object of the present invention to provide for alternative regulatory elements suitable for recombinant production methods, which are simple and efficient.